Late amniocentesis with uniparental disomy testing following successful in vitro fertilization and transfer of three mosaic embryos in a pregnancy with a favorable outcome.

OBJECTIVE: We present late amniocentesis with the application of uniparental disomy (UPD) testing following successful in vitro fertilization (IVF) and transfer of three mosaic embryos in a pregnancy with a favorable outcome. CASE REPORT: A 41-year-old, gravida 2, para 0, woman underwent late amniocentesis at 28 weeks of gestation because of advanced maternal age. The pregnancy was conceived by IVF and transfer of three mosaic embryos, i.e., one embryo with mosaic trisomies 20 and 2, one embryo with mosaic partial trisomies 9 and 3 and one embryo with partial trisomies 11 and 17. First-trimester non-invasive prenatal testing (NIPT) and fetal ultrasound revealed no abnormal findings. Late amniocentesis revealed a karyotype of 46,XY. Array comparative genomic hybridization (aCGH) revealed the result of arr [GRCh37] (X,Y) × 1, (1-22) × 2. Polymorphic DNA marker analysis using the DNAs extracted from the uncultured amniocytes and parental bloods excluded UPD 20. At 38 weeks of gestation, a healthy 3010-g male baby was delivered with no phenotypic abnormalities. CONCLUSION: Prenatal diagnosis of normal karyotype following mosaic embryo transfer should include UPD testing if necessary.

Killer cell immunoglobulin-like receptors (KIR) and human leukocyte antigen-C (HLA-C) allorecognition patterns in women with endometriosis.

Endometriosis shares similarities with several autoimmune diseases. The human leukocyte antigen (HLA)-C genotype is associated with several human autoimmune diseases. HLA-C is a ligand of killer cell immunoglobulin receptors (KIRs) and is an essential regulator of natural killer cell activity, which is associated with endometriosis progression. Polymorphisms in HLA-C and KIR affect the activity of NK cells and susceptibility to several diseases. Therefore, we attempted to investigate an association between HLA-C genotype and KIR polymorphism and the occurrence of endometriosis. We tested the association of certain KIR and HLA-C combinations and the development of endometriosis by characterizing both KIR and HLA-C genes in 147 women with endometriosis and 117 controls. The HLA-C genotypes and KIR polymorphisms were analyzed via DNA-based method for higher-resolution genotyping. We found that the occurrence of HLA-C*03:03*01 was increased in endometriosis than in control groups. Analysis of various KIR haplotypes revealed differences between the endometriosis and control cohorts. The number of KIR centromeric A/A haplotypes was increased in the endometriosis group than controls. Moreover, the endometriosis cohort was characterized by reduced number of KIR2DS2-positive individuals in the Han Chinese population. Our current findings suggest that the KIR and HLA-C genotypes are associated with the pathogenesis of endometriosis.

Adjusted mitochondrial DNA quantification in human embryos may not be applicable as a biomarker of implantation potential.

OBJECTIVE: To evaluate the feasibility of adjusted mitochondrial DNA quantification in human embryos as a biomarker for implantation potential. DESIGN: Double-blind, observational, prospective analysis of an Asian population in a single university-affiliated in vitro fertilization center. A total of 1617 embryos derived from 380 infertile couples were collected. The DNA from blastomere biopsy (n = 99) or trophectoderm biopsy (n = 1518) were analyzed with next-generation sequencing. RESULTS: The adjusted mtDNA quantification followed a non-normal distribution in both types of the embryos. When stratified by ploidy status, the adjusted mtDNA quantification was significantly higher in aneuploid trophectoderm than in euploid cells, but not in blastomeres. The adjusted mtDNA quantification of embryos showed significant but very weak positive correlation in total trophectoderm cells with maternal age (Spearman's correlation, r = 0.095, p = 0.0028) but neither in blastomeres nor stratified by ploidy status. The median adjusted mtDNA quantification was also significantly higher in aneuploid blastocysts than in euploid ones while corrected with embryo morphology. Viable embryos did not contain significantly different quantities of adjusted mtDNA compared with nonviable embryos (implanted n = 103, non-implanted n = 164; median 0.00097 vs. 0.00088, p = 0.21) in 267 transferred blastocysts. CONCLUSION: Quantification of adjusted mitochondria DNA in human embryos was significantly lower in euploid blastocysts than in aneuploid blastocysts. However, no statistically significant differences regarding implantation outcome were evident. To our best knowledge, this study provides the largest scale and the first correlation data between mitochondria copy number and human embryo implantation potential in Asians.

Early blastulation (EB) of day 4 embryo is predictive of outcomes in single embryo transfer (SET) cycles.

OBJECTIVE: The aim of this study was to assess whether the early blastulation (EB) of day 4 embryo is a useful predictor for outcomes in fresh elective single embryo transfer (eSET) cycles. MATERIALS AND METHODS: We retrospectively enrolled patients undergoing fresh SET cycles in our hospital from April 2014 to September 2016 and met with the following criteria: 1) age <38 years, 2) first IVF/ICSI cycle, 3) at least two blastocysts with morphological grading better than or equal to 4BB. RESULTS: A total of 81 patients were included. Of whom, 55 patients (68%) had undergone eSET with embryos that had early blastulation on day 4 while the other 26 patients had had no EB. Early blastulation has shown a higher rate of good blastocyst (84.3% vs. 60.5%, p < 0.0001). The clinical pregnancy rate of EB group was significantly higher than that of non-EB group (56.4% vs. 27.0%, p = 0.013). There is also a tendency in EB group to have a lower abortion rate (3.23% vs. 28.6%, p = 0.081). CONCLUSIONS: EB on day 4 is a useful predictor of the quality of the following embryos (i.e. day 5 embryo). It is a simple tool in selecting the best embryo to get a higher pregnancy rate in fresh eSET cycles. TRIAL REGISTRATION: This study was supplementally registered by the MacKay Memorial Hospital Institutional Review Board on April 18, 2017 (registration No. 17MMHIS039e).

Outcomes of anti-Müllerian hormone-tailored ovarian stimulation protocols in in vitro fertilization/intracytoplasmic sperm injection cycles in women of advanced age.

OBJECTIVE: We aimed to compare the outcomes of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatments in women of advanced age (>40 years) using anti-Müllerian hormone (AMH)-tailored ovarian stimulation protocols versus conventional protocols based on antral follicle count (AFC). MATERIALS AND METHODS: We retrospectively reviewed 210 women who underwent IVF/ICSI cycles: 116 women underwent stimulation protocols that were tailored to their AMH levels, whereas 94 women received treatment using conventional stimulation protocols based on AFC as the ovarian reserve marker. RESULTS: The following parameters were significantly higher in the AMH-tailored group than in the conventional group: initial and total doses (IU) of recombinant follicle-stimulating hormone (rFSH) used for stimulation (514.2 ± 137.9 vs. 452.3 ± 135.3, p = 0.001; 4713.8 ± 1618.8 vs. 4047.2 ± 1366.0, p = 0.007, respectively), ovum pick-up rate (OPU; 88.8% vs. 75.5%, p = 0.016), serum estradiol (E2) level on the day of human chorionic gonadotropin (hCG) administration (1818.5 ± 1422.4 vs. 1394.0 ± 929.0 pg/mL, p = 0.028), number of oocytes retrieved (7.4 ± 5.1 vs. 5.5 ± 3.4, p = 0.007), number of embryos per case (4.2 ± 3.2 vs. 3.3 ± 2.5, p = 0.048), clinical pregnancy rates (22.4% vs. 8.5%, p = 0.008), implantation rates (13.1% vs. 3.9%, p = 0.001), and live birth rates per cycle (15.5% vs. 6.4%, p = 0.049). CONCLUSION: Individualized controlled ovarian stimulation (COS) protocols tailored to patients' AMH levels may improve the pregnancy rate, implantation rate, and live birth rate in women of advanced age undergoing IVF/ICSI compared with those receiving conventional stimulation protocols.

A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented.

Treatment of infertile women with unilateral tubal occlusion by intrauterine insemination and ovarian stimulation.

OBJECTIVE: The aim of this study was to evaluate the value of intrauterine insemination (IUI) combined with ovarian stimulation in women with unilateral tubal occlusion detected on hysterosalpingography (HSG). MATERIALS AND METHODS: A total of 703 patients undergoing IUI and controlled ovarian hyperstimulation were enrolled in this study. The study group consisted of 133 patients treated for unilateral tubal occlusion diagnosed by HSG during 2005-2011. The control group consisted of 570 patients with unexplained infertility treated during the same period. In all cases of the retrospective study, menstrual cycles were regular, basal serum follicle-stimulating hormone levels and sperm parameters were normal. RESULTS: There were no significant differences in pregnancy rate per cycle between the study (17.3%) and control groups (18.9%). The pregnancy rate was higher in patients with proximal tubal occlusion (21.7%) compared with mid-distal tubal occlusion (12.5%) or unexplained infertility (18.9%), but the difference was not statistically significant. CONCLUSIONS: Infertile patients with only unilateral proximal tubal occlusion detected on HSG can be treated initially by IUI combined with ovarian stimulation. The cycle outcomes in patients with proximal tubal occlusion are similar to patients with unexplained infertility. However, the stimulated IUI might not be a good choice for patients with unilateral mid-distal tubal occlusion because of a lower success rate, although further evidence is needed.

Dual trigger with combination of gonadotropin-releasing hormone agonist and human chorionic gonadotropin significantly improves the live-birth rate for normal responders in GnRH-antagonist cycles.

OBJECTIVE: To investigate whether dual triggering of final oocyte maturation with a combination of gonadotropin-releasing hormone (GnRH) agonist and human chorionic gonadotropin (hCG) can improve the live-birth rate for normal responders in GnRH-antagonist in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) cycles. DESIGN: Retrospective cohort study. SETTING: Infertility unit of a university-affiliated medical center. PATIENT(S): Normal responders to controlled ovarian hyperstimulation who were undergoing IVF-ICSI with a GnRH antagonist protocol. INTERVENTION(S): Standard dosage of hCG trigger (6,500 IU of recombinant hCG) versus dual trigger (0.2 mg of triptorelin and 6,500 IU of recombinant hCG). MAIN OUTCOME MEASURE(S): Live-birth, clinical pregnancy, and implantation rates per cycle. RESULT(S): A total of 376 patients with 378 completed cycles with embryo transfer were enrolled (hCG trigger/control group: n = 187; dual trigger/study group: n = 191). The dual trigger group demonstrated statistically significantly higher implantation (29.6% vs. 18.4%), clinical pregnancy (50.7% vs. 40.1%), and live-birth (41.3% vs. 30.4%) rates as compared with the hCG trigger group. There was no statistically significant difference in terms of patient demographics, cycle parameters, or embryo quality. CONCLUSION(S): Dual trigger of final oocyte maturation with a GnRH-agonist and a standard dosage of hCG in normal responders statistically significantly improves implantation, clinical pregnancy, and live-birth rates in GnRH-antagonist IVF cycles.

Comparison of the clinical outcomes between fresh blastocyst and vitrified-thawed blastocyst transfer.

OBJECTIVE: To compare the clinical outcomes between fresh and vitrified-thawed day 5 blastocyst transfers. DESIGN: Retrospective case control study. SETTING: Tertiary referral center. PATIENT(S): Patients 38 years of age or less who underwent IVF/ICSI cycles with fresh or frozen-thawed blastocysts transferred from June 1, 2009 to November 30, 2011 INTERVENTION(S): Vitrification and thawing of day 5 blastocysts using the Cryotop method. (Kitazato BioPharma Co., Ltd., Fuji city, Shizuoka, Japan) MAIN OUTCOME MEASURE(S): Clinical pregnancy rate, implantation rate, ongoing pregnancy rate, and multiple pregnancy rates. RESULTS: Of the 118 cycles in the fresh transfer group, 234 blastocysts were transferred. The clinical pregnancy rate was 66.1 % and implantation rate was 50.9 %. The ongoing pregnancy rate was 56.8 % and the rates for singleton and twin pregnancies were 53.7 % and 44.8 %. Of the 59 cycles in the vitrified-thawed group, 111 blastocysts were transferred. The clinical pregnancy rate was 59.3 % and implantation rate was 43.2 %. The ongoing pregnancy rate was 47.5 % and the rates for singleton and twin pregnancies were 60.7 % and 39.3 %. The clinical pregnancy rate, implantation rate and ongoing pregnancy rate did not differ significantly between the two groups. CONCLUSIONS: The implantation rates were not significantly different between the fresh and the vitrified-thawed groups. Thus, single embryo transfer may be considered in fresh cycles to decrease multiple pregnancy rates. The surplus embryos should be vitrified for the frozen embryo transfer to improve the cumulative pregnancy rate.

The predictability of serum anti-Müllerian level in IVF/ICSI outcomes for patients of advanced reproductive age.

BACKGROUND: The role of serum anti-Müllerian hormone (AMH) as predictor of in-vitro fertilization outcomes has been much debated. The aim of the present study is to investigate the practicability of combining serum AMH level with biological age as a simple screening method for counseling IVF candidates of advanced reproductive age with potential poor outcomes prior to treatment initiation. METHODS: A total of 1,538 reference patients and 116 infertile patients aged greater than or equal to 40 years enrolled in IVF/ICSI cycles were recruited in this retrospective analysis. A reference chart of the age-related distribution of serum AMH level for Asian population was first created. IVF/ICSI patients aged greater than or equal to 40 years were then divided into three groups according to the low, middle and high tertiles the serum AMH tertiles derived from the reference population of matching age. The cycle outcomes were analyzed and compared among each individual group. RESULTS: For reference subjects aged greater than or equal to 40 years, the serum AMH of the low, middle and high tertiles were equal or lesser than 0.48, 0.49-1.22 and equal or greater than 1.23 ng/mL respectively. IVF/ICSI patients aged greater than or equal to 40 years with AMH levels in the low tertile had the highest cycle cancellation rate (47.6%) with zero clinical pregnancy. The nadir AMH level that has achieved live birth was 0.56 ng/mL, which was equivalent to the 36.4th percentile of AMH level from the age-matched reference group. The optimum cut-off levels of AMH for the prediction of nonpregnancy and cycle cancellation were 1.05 and 0.68 ng/mL, respectively. CONCLUSIONS: Two criteria: (1) age greater than or equal to 40 years and (2) serum AMH level in the lowest tertile (equal or lesser than 33.3rd percentile) of the matching age group, may be used as markers of futility for counseling IVF/ICSI candidates.

Outcomes of high initial daily doses of gonadotropin in patients with poor ovarian reserve.

OBJECTIVE: To evaluate the in vitro fertilization and intracytoplasmic sperm injection outcomes after high initial doses of follicle-stimulating hormone (FSH) in patients with poor ovarian reserve. MATERIALS AND METHODS: For in vitro fertilization/intracytoplasmic sperm injection patients younger than 40 years of age, 345 cycles were examined from April 2003 to April 2007. As a control, 218 cycles received gonadotropin-releasing hormone agonist and regular initial doses of FSH from day 3 of the treated cycle. The remaining 127 cycles were treated with high initial doses of FSH with an antagonist or low doses of gonadotropin-releasing hormone because of poor ovarian reserve. RESULTS: When higher initial doses of FSH were used, lower estradiol levels on the day of human chorionic gonadotropin injection and less mature oocytes were retrieved from the group with poor ovarian reserve. Clinical pregnancy rates per embryo transfer were similar (45.7%vs. 48.2%, p = 0.686). There was a trend of lower ongoing pregnancy rate per cycle (28.3%vs. 38.5%, p = 0.05) in the study compared with the control group. In the subgroups with high doses of FSH, neither protocol was superior in terms of clinical (45.5%vs. 46.2%, p=0.952) or ongoing pregnancy rates per embryo transfer (37.9%vs. 42.3%, p=0.695). CONCLUSION: There was no significant difference in clinical pregnancy rate of the two groups when good embryos were obtained. The group with poor ovarian reserve had lower ongoing pregnancy rates per cycle. For patients with expected poor ovarian response, treatment with high doses of FSH initially is an option.

Increased expression of platelet-derived growth factor C messenger ribonucleic acid in uterine leiomyomata.

This study was intended to investigate the possible roles of the platelet-derived growth factor (PDGF) gene family's involvement in the pathogenesis of uterine leiomyomata. We examined the differential gene expressions of PDGF-A, -B, -C, -D, PDGF receptor alpha (PDGFR-alpha), and receptor beta (PDGFR-beta) between uterine leiomyomata and the adjacent normal myometrium. Expression of PDGF-C in leiomyomata was significantly higher (approximately 2.4-fold) than in the adjacent normal myometrium, whereas there were no significant differences in the expressions of PDGF-A, -B, -D, PDGFR-alpha, or PDGFR-beta between leiomyomata and the adjacent myometrial tissues.

Female infertility and disrupted angiogenesis are actions of specific follistatin isoforms.

The circulating and tissue-bound forms of follistatin (FST315 and FST288, respectively) modulate the actions of activins. FST knockout (KO/null) mice, lacking both isoforms, die perinatally with defects in lung, skin, and the musculoskeletal system. Using constructs of the human FST gene engineered to enable expression of each isoform under the control of natural regulatory elements, transgenic mouse lines were created and crossed with FST null mice to attempt to rescue the neonatal lethality. FST288 expression alone did not rescue the neonatal lethality, but mice expressing FST315 on the KO background survived to adulthood with normal lung and skin morphology and partial reversal of the musculoskeletal defects noted in FST KO mice. The FST315 rescue mice displayed a short period of neonatal growth retardation, impaired tail growth, and female infertility. The latter may be due to failure of corpus luteum formation, a decline in the ovarian follicular population, and an augmented uterine inflammatory response to mating. Failure of corpus luteum formation and impaired tail growth indicate abnormal vascularization and suggest that FST288 is required for the promotion of angiogenesis. The augmented uterine inflammatory response may result from the failure of FST315 to modulate the proinflammatory actions of activin A in the uterus or may result from the altered steroid milieu associated with the ovarian abnormalities. Although we cannot definitively conclude that the remaining defects are due to the absence of a particular isoform or due to variable expression of each, these models have demonstrated novel physiological processes that are influenced by FST.

Early cleavage does not predict treatment outcome following the use of GnRH antagonists in women older than 35.

OBJECTIVE: To compare the early-cleavage rates and the implantation potential of embryos between the GnRH antagonist and agonist long stimulation protocols in women older than 35 years. DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENTS: Two hundred twenty patients older than 35 years old underwent IVF. INTERVENTION(S): Sixty-eight patients received GnRH antagonist protocol (GnRH antagonist group) and 152 patients received GnRH agonist long stimulation protocol (GnRH agonist group). MAIN OUTCOME MEASURE(S): Early-cleavage rate of zygotes, implantation rate, and pregnancy rate. RESULT(S): Early-cleavage rate of zygotes was significantly lower in the GnRH antagonist group than agonist group (21.8% vs. 32.6%, P<.0001). In the GnRH antagonist group, the pregnancy rate was not significantly different between the early-cleavage and late-cleavage subgroups (40.0% vs. 47.4%). In the GnRH agonist group, the pregnancy rate was significantly higher in the early-cleavage subgroup than in the late-cleavage subgroup (61.0% vs. 29.8%, P<.0001). CONCLUSION(S): In women older than 35 years, the early-cleavage rate of zygotes is significantly lower in the GnRH antagonist group than the agonist group. Early-cleavage status of zygotes is not a reliable predictor for embryo implantation in patients receiving the GnRH antagonist protocol.

The role of activin, follistatin and inhibin in testicular physiology.

The role of the inhibins, activins and follistatins in testicular function are being more clearly defined following studies describing the cellular localisation of these proteins to the testis and the availability of specific assay systems enabling measurement of these proteins. Taken together with the results of targetted gene inactivation experiments, several concepts emerge. Inhibin B is predominantly produced by the Sertoli cell in many adult male mammals whereas there is a perinatal peak of inhibin A in the rat. In contrast, activin A has its highest concentrations in the immediate post-natal period during which it is involved in the developmental regulation of both germ cells and Sertoli cells being modulated by follistatin. Activin A levels are considerably lower in the adult testis but Sertoli cell production is stimulated by interleukin-1 and inhibited by FSH. Little is known about the production of activin B due to the absence of a suitable assay but the beta(B) subunit mRNA is expressed in germ cells and Sertoli cells and is stage-dependent. This pattern of expression suggest that it may be involved in autocrine or paracrine actions within the seminiferous epithelium.

Regulation of ovarian function by the TGF-beta superfamily and follistatin.

The role of follistatin as an activin-binding protein has dominated the study of this molecule for the last 10 years. However, there is emerging evidence that follistatin has a role in modulating the biology of other members of the transforming growth factor beta (TGF-beta) superfamily. This review summarizes the current concepts encompassing follistatin biochemistry as well as molecules with which it is functionally associated. Moreover, the importance of the two follistatin isoforms (follistatin-288 and follistatin-315) is discussed with particular emphasis on the regulation of the ovary. In addition to activin, this review discusses the functions of other members of the TGF-beta superfamily, for example growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), BMP-6, BMP-4 and BMP-7, in the ovary, and the potential interactions between follistatin and these growth factors. The complex network of TGF-beta superfamily growth factor members involved in the modulation of ovarian function and the interactions of follistatin with these proteins is highlighted.